U.S. patent application number 10/539437 was filed with the patent office on 2006-10-19 for method and device for pcr-amplification and detection of nucleotide sequences.
Invention is credited to Walter Gumbrecht, Manfred Stanzel.
Application Number | 20060234236 10/539437 |
Document ID | / |
Family ID | 32477844 |
Filed Date | 2006-10-19 |
United States Patent
Application |
20060234236 |
Kind Code |
A1 |
Gumbrecht; Walter ; et
al. |
October 19, 2006 |
Method and device for pcr-amplification and detection of nucleotide
sequences
Abstract
A DNA-Chip includes a flat carrier and an array of spots
containing probe molecules (oligonucleotides) which are arranged on
said carrier. Each spot is associated with a microelectrode
arrangement for impedance spectroscopic detection of binding events
occurring between the probe molecules and target molecules (DNA
fragments) applied by way of an analyte solution. In order to
increase the sensitivity or the binding specific measuring effects
of the biochip, the electrode arrangement is at least partially
embedded in a hydrophilic reaction layer containing probe molecules
and which is permeable to target molecules.
Inventors: |
Gumbrecht; Walter;
(Herzogenaurach, DE) ; Stanzel; Manfred;
(Erlangen, DE) |
Correspondence
Address: |
HARNESS, DICKEY & PIERCE, P.L.C.
P.O.BOX 8910
RESTON
VA
20195
US
|
Family ID: |
32477844 |
Appl. No.: |
10/539437 |
Filed: |
December 15, 2003 |
PCT Filed: |
December 15, 2003 |
PCT NO: |
PCT/DE03/04136 |
371 Date: |
March 20, 2006 |
Current U.S.
Class: |
435/6.11 ;
427/2.11; 435/287.2; 435/91.2 |
Current CPC
Class: |
C12Q 1/686 20130101;
C12Q 1/686 20130101; C12Q 2565/501 20130101; C12Q 2565/607
20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 435/287.2; 427/002.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34; C12M 1/34 20060101
C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 2002 |
DE |
10259819.3 |
Claims
1. A method for PCR amplification and detection of nucleotide
sequences, comprising: using an array of a plurality of microspots
forming analytical positions, said microspots including as probe
molecule at least one immobilized oligonucleotide which is
hybridizable with a target sequence to be identified of a DNA
fragment; applying an analyte solution including PCR reagents and a
plurality of target sequences to the microspots in such a way that
it completely covers the array; subjecting the array to a
thermocycling process to amplify the target sequences; and
detecting hybridization events on probe molecules immobilized at
one analytical position with the aid of a microelectrode
arrangement.
2. The method as claimed in claim 1, wherein a hydrophilic reaction
layer having coupling groups for covalent binding of probe
molecules is used.
3. The method as claimed in claim 2, wherein the reaction layer
used is a hydrogel.
4. The method as claimed in claim 2, wherein a free-radically
crosslinkable hydrogel based on at least one of acrylamide with
maleic anhydride and glycidyl (meth)acrylate as coupling groups is
used.
5. The method as claimed in claim 1, wherein a biochip including a
semiconductor layer and an insulating layer connected therewith is
used, the side of the insulating layer, which faces away from the
semiconductor layer, carrying the electrode arrangement and the
reaction layer.
6. The method as claimed in claim 5, wherein the semiconductor
layer used is a silicon layer.
7. The method as claimed in claim 1, wherein an analyte solution is
used which includes an external primer pair.
8. The method as claimed in claim 1, wherein an analyte solution is
used which includes a plurality of DNA fragments having a different
target sequence and a single external primer pair suitable for the
amplification of all target sequences.
9. The method as claimed in claim 1, wherein an analyte solution is
used which includes an external primer acting together with the one
strand of at least one DNA fragment and in that a counter strand is
elongated within a reaction layer with the aid of an internal
primer immobilized there.
10. The method as claimed in claim 1, wherein an analyte solution
is used in which an internal primer pair specifically hybridizing
with a target sequence is immobilized in a microspot.
11. A device for carrying out the method as claimed in claim 1,
comprising a biochip having an array of microspots which form
analytical positions and which are covered by a hydrophilic
reaction layer.
12. The device as claimed in claim 11, wherein the biochip with
hydrophilic reaction layer is arranged in a housing having an
opening for an analyte solution.
13. The device as claimed in claim 11, wherein the biochip contains
carriers for the microspots as substrate.
14. The device as claimed in claim 11, wherein the substrate
consists of a semiconductor material, to which an insulating layer
has been applied.
15. The device as claimed in claim 11, wherein the biochip is a
prefabricated silicon chip having thin-layer microelectrodes
implemented therein.
16. The method as claimed in claim 3, wherein a free-radically
crosslinkable hydrogel based on at least one of acrylamide with
maleic anhydride and glycidyl (meth)acrylate as coupling groups is
used.
17. The method as claimed in claim 1, wherein an analyte solution
is used which includes a primer pair which hybridizes with a target
DNA outside a target sequence.
18. The method as claimed in claim 1, wherein an analyte solution
is used which includes an external primer acting together with the
one strand of at least one DNA fragment and in that a counter
strand is elongated within a reaction layer with the aid of a
primer which specifically hybridizes with the target sequence,
immobilized there.
19. The device as claimed in claim 11, wherein the substrate
consists of silicon, to which an insulating layer has been applied.
Description
[0001] This application is a PCT National Stage Application of
PCT/DE2003/004136 filed Dec. 15, 2003, which claims priority under
on German Patent Application No. DE 102 59 819.3 filed in Germany
on Dec. 19, 2002, the entire contents of which is hereby
incorporated herein by reference.
FIELD
[0002] The invention generally relates to a method for PCR
amplification and detection of nucleotide sequences. A method of
this kind serves, for example in medical diagnostics, to track down
infectious target sequences of viral or bacterial DNA. In addition,
the invention also generally relates to a corresponding device for
carrying out the method.
BACKGROUND
[0003] During a PCR (Polymerase Chain Reaction), the sample to be
investigated is subjected to a cyclical temperature treatment in
which the DNA fragments are essentially duplicated with the aid of
a primer pair and a polymerase. For this kind of analyses, there
are nowadays processes available in which the PCR is carried out on
a microchip which has an array of microspots which form "gel pads"
(WO 01/34842 A2). In order to enable hybridizations within the
microspots to be detected by fluorescence spectroscopy, the known
processes involve adding a labeled primer to the analyte
solution.
[0004] Methods for amplifying and detecting nucleic acids are known
from the prior art. Here, gel pads may form separate microspots as
hydrophilic reaction layers on a microarray, said gel pads
containing oligonucleotides which can hybridize with target nucleic
acids to be identified. Furthermore, the printed publication
"Nucleic Acids Res." (1999) 27 (18) e19, pages 1 to 6, discloses
carrying out amplification reactions in gel pads on a microarray
and detection reactions by way of single base elongation. In this
connection, mention should further be made of DE 196 10 115 C2
which discloses an array having a microelectrode arrangement and of
WO 01/42508 A2 which discloses gel pads with immobilized probes in
contact with microelectrodes.
[0005] Finally, WO 99/36576 A1 involves the use of gel pads in an
array and also methods and systems for their preparation, it being
intended to prepare "intelligent gels" as reaction layers.
SUMMARY
[0006] It is an object of an embodiment of the invention to propose
an improved method for amplification and detection of nucleotide
sequences, which makes possible continuous monitoring of the PCR
and, in particular, simultaneous investigation of a plurality of
target sequences or a plurality of mutations of a target sequence
in a simple manner. In addition, it is intended to produce a device
which makes possible, in particular, an electrochemical
measurement.
[0007] The method of an embodiment of the invention includes the
following: [0008] a) providing a microchip having an array of a
plurality of microspots forming analytical positions, which in each
case comprise a hydrophilic reaction layer and a micro-electrode
arrangement embedded therein, said reaction layer comprising as
probe molecule at least one immobilized oligonucleotide which is
hybridizable with a target sequence to be identified of a DNA
fragment, [0009] b) applying an analyte solution comprising a
plurality of target sequences and PCR reagents to the microchip in
such a way that it completely covers the array, [0010] c)
subjecting the array to a thermocycling process in order to amplify
the target sequences, [0011] d) detecting hybridization events on
probe molecules immobilized at one analytical position with the aid
of the microelectrode arrangement assigned to said position.
[0012] This method of at least one embodiment has first of all the
advantage that it is possible to detect binding or hybridization
events in a microspot from the start of the PCR, without
interruption of the ongoing reaction cycles and with minimum
equipment. Since the microspots contain electrode arrangements
which are independent of one another, each analytical position can
be addressed individually and thus correlated to a particular probe
molecule or a desired target sequence.
[0013] It is therefore possible to monitor a hybridization in a
multiplicity of microspots in a very simple manner at the same
time. In contrast, optical read out would require an optical
recording system which is technically complex, if only due to the
small size of the spots and their arrangement in a very narrow
space. The technical complexity becomes even greater, if arrays
having a large number of microspots are to be read out.
[0014] Owing to their electric partial charges, the nucleotide
sequences held in a microspot by hybridization with immobilized
probe molecules alter electrical parameters such as, for example,
the conductance within a microspot or the impedance of an electrode
arrangement. This makes possible an electrochemical or electrical
evaluation using a device of an embodiment of the invention
including a biochip with microelectrode arrangement.
[0015] DE 196 10 115 C2 discloses a biochip which can be read out
impedance-spectroscopically and which already contains a plurality
of interdigital electrode arrangements on a carrier, with probe
molecules being immobilized on the electrodes and on the surfaces
located between said electrodes. However, this kind of detecting
binding events has the problem that the dimensions of the electrode
structures differ from molecular dimensions by orders of
magnitude.
[0016] It is possible, with still justifiable technical complexity,
to prepare electrodes which have a width of between 1 and 10 .mu.m,
in particular of about 5-10 .mu.m, are at a distance of the same
size and have a thickness of from about 0.1 to 0.5 .mu.m. The
impedance-spectroscopically recordable range of the electric field
of such an electrode arrangement extends from about 5 to 10 .mu.m
beyond the carrier surface or the plane formed by the electrode
arrangement. In contrast, a probe molecule having, for example, 100
base pairs has a length of only about 30 nm, i.e. 0.3 .mu.m. The
influence of binding events in a monomolecular layer of probe
molecules, immobilized on the sensor area or the electrodes, on the
electric field or on the impedance of the electrode arrangement is
correspondingly low.
[0017] Due to the fact that, according to an embodiment of the
invention, the electrode arrangement is embedded at least partially
in a hydrophilic reaction layer containing probe molecules and
permeable for target molecules, it is possible to collect within
the reaction layer a much higher number of probe molecules or
target sequences than in a monomolecular layer. This results in a
much larger influence of the electric field or of the
impedance-spectroscopic recording range of the electrode
arrangement.
[0018] A biochip designed in this way has a correspondingly higher
measuring sensitivity. In contrast, in the case of conventional
biochips, an increase in the concentration of the target sequences,
obtained by PCR, would not result in an increase in sensitivity,
owing to the small supply of probe molecules.
[0019] The reaction layer used in the method of an embodiment of
the invention must be thermally stable to about 95.degree. C. in
order to carry out a PCR. Thermally stable here means that the
composition of the reaction layer is even at the temperature
indicated such that it holds onto probe molecules, that
hybridization/denaturation (melting) of target sequences and probe
molecules can take place in it unimpededly and that it also
essentially retains its other properties. For immobilization, the
reaction layer preferably contains polymers with coupling groups to
which probe molecules are covalently bound. This guarantees for
sure that binding pairs of target sequences and probe molecules are
retained in the reaction layer during rinsing processes following a
PCR.
[0020] A particularly suitable reaction layer includes or even
consists of a hydrogel. Hydrogels form an aqueous environment in a
mechanically stable form, which permits mass transfer with a
predominantly aqueous analyte. Free-radically crosslinkable
hydrogels based on acrylamide, with maleic anhydride and/or
glycidyl (meth)acrylate as coupling groups, have proved
particularly suitable.
[0021] In a further preferred embodiment, the flat carrier of the
biochip includes silicon (Si) as substrate and an insulating layer
connected therewith, the side of the latter, which faces away from
the silicon layer, carrying the electrode arrangement and the
reaction layer. Such an arrangement enables the electrical
interconnection of the electrode arrangement to be implemented
using the technology known from Si memory chips.
[0022] A particular advantage of the proposed method is the fact
that the method permits a larger variety of different possible
designs in the case of simultaneous or multiplex studies. The
reason for this is, inter alia, that it is not necessary to
incorporate a label into amplicons produced during the PCR, which,
especially in complex tests, holds the risk of undesired
interactions arising between the substances required for labeling
and between these and target sequences to be identified.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Further details and advantages of the invention are obtained
from the description of the figures below of example embodiments on
the basis of the drawings. In the figures:
[0024] FIG. 1 depicts a simplified perspective representation of a
microchip comprising a flat carrier and an array of microspots,
[0025] FIG. 2 depicts a cross section through a spot according to
line II-II in FIG. 1, as an enlarged detail,
[0026] FIG. 3 depicts a detail of an electrode arrangement assigned
to a spot,
[0027] FIG. 4 depicts an embodiment of a microchip having a 4-pole
electrode arrangement in a representation corresponding to FIG.
2,
[0028] FIG. 5 depicts the electrode arrangement of the microchip of
FIG. 4 in a representation corresponding to FIG. 3,
[0029] FIG. 6 depicts a diagrammatic representation which
illustrates a first method variant of a PCR-assisted analysis,
[0030] FIG. 7 depicts a diagrammatic drawing which indicates the
mode of action of an unspecific primer pair,
[0031] FIG. 8 depicts a diagrammatic representation of a
modification of the first method variant,
[0032] FIG. 9 depicts a diagrammatic representation of a second
method variant,
[0033] FIG. 10 depicts a diagrammatic representation of a third
method variant.
DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS
[0034] As FIG. 1 indicates, an element referred to as biochip 1
includes a flat carrier 2 to one side of which a spot array 3 has
been applied. A microspot referred to hereinbelow as spot 4
contains immobilized probe molecules, for example oligonucleotides.
If an analyte solution containing unknown target molecules is
applied to a spot 4, then the target molecule couples to the probe
molecule, if the base sequences correspond to one another. The
property change caused by such a binding event, for example changes
in the specific resistance, the impedance or the dielectricity
constant, can be recorded by an electrode arrangement 5.
[0035] The spot array 3 or microchip 1 with electrodes implemented
therein forms a device which permits online monitoring. Such a
device may have different electrode arrangements which are depicted
in FIGS. 3 and 5. FIGS. 2 and 4, in contrast, depict the
phenomenology of immobilization and measurement in such
arrangements.
[0036] The example embodiment of FIG. 2 contains a two-pole
electrode arrangement. The latter has been applied, for example,
with the aid of a photolithographic process on the flat carrier 2.
The electrode arrangement 5 includes two electrodes 6, 7 which have
the form of an interdigital structure, i.e. each electrode includes
a plurality of strip-like partial electrodes 6a, 7a parallel to one
another, which extend in each case into the space between two
partial electrodes of the in each case other electrode. The partial
electrodes 6a, 7a are connected to one another by a likewise
strip-like connecting conductor 6b, 7b which extends at an angle to
the partial electrodes 6a, 7a.
[0037] A high-frequency alternating current in the megahertz range
is applied to the electrodes 6, 7. The width 8 of the partial
electrodes 6a, 7a is approx. 1 .mu.m, their height 9 is from about
100 to 500 nm. The distance 10 between the partial electrodes 6a,
7a is likewise approx. 1 .mu.m.
[0038] The flat carrier 2 includes a silicon layer 12 and an
insulating layer 13, including or even consisting of a polymer and
arranged between said silicon layer and the electrodes 6, 7. The
electrical interconnections and parts required, for example for an
impedance-spectroscopic measurement of binding events, are
implemented in the usual way by way of an appropriate topology of
the silicon layer, and this is not shown in FIG. 2 in any
detail.
[0039] A reaction layer 14 composed of a hydrogel which will be
described in more detail below has been applied to the insulating
layer 13. It may be expedient to provide the flat carrier 2 or the
silicon layer 13 in the region of a spot with a depression filled
with the reaction layer 14 (see FIGS. 6, 8-10). Probe molecules 15
are embedded and homogeneously distributed in the reaction layer 14
or the hydrogel, and this is depicted in FIG. 2 in an enlarged and
symbolic manner. A probe molecule of 300 bases has a length of
about 100 nm. Consequently, a unimolecular layer of probe molecules
in conventional microchips has a thickness which at most
corresponds about to the line 16 in FIG. 2.
[0040] It is readily understood that such a layer can take
relatively few probe molecules 15 and, correspondingly, can
influence the electric field of the electrode arrangement only
slightly in the case of binding or hybridization events. In
contrast to this, the reaction area in a microchip of the
invention, which area contains probe molecules 15 and is penetrated
by field lines 17, is substantially enlarged and offers space for a
several powers of ten higher number of probe molecules 15. If an
analyte solution 18 is applied to a spot array 3 designed in this
way or to a spot 4, then the target molecules 19 or target
sequences contained therein and depicted in FIG. 2 likewise on an
exaggerated scale and only symbolically find a substantially larger
number of possible binding partners in the form of the probe
molecules 15.
[0041] The dimensions of the reaction layer 14 and its thickness,
for example from 5 to 10 .mu.m, are preferably such that the
impedance-spectroscopic recording range is basically completely
utilized, this being the case at a thickness of the reaction layer
of from about 5 to 10 .mu.m. It is thus possible, at an appropriate
concentration of probe molecules 15 in this range, to increase the
binding-specific measurement effect of the microchip substantially.
The composition of the reaction layer is such that it provides an
aqueous reaction medium. It is furthermore such that target
molecules 19 or else other substances required for a reaction, for
example polymerase, can diffuse into said layer, without their
reactivity being impaired in the process.
[0042] As already mentioned above, the reaction layer 14 used
according to an embodiment of the invention is a hydrogel. A
hydrogel is an aqueous environment in a mechanically stable form
with simultaneous guarantee of mass transfer in a predominantly
aqueous surrounding. It is possible, by choosing the chemical
composition, with respect to the components and the ratios between
them, to vary the properties of the hydrogels, such as water
content, swelling behavior, mechanical stability, etc., over a wide
range.
[0043] A hydrogel, which can be easily prepared and which has good
adhesion both to the electrode arrangement 5 and to the insulating
layer 13, is a free-radically crosslinkable hydrogel based on
acrylamide, which contains a comonomer which enables
correspondingly modified probe molecules to be covalently coupled
via linker groups. The hydrogel includes, in addition to the
monomeric precursor of the polyacrylamide, a crosslinker, at least
one free radical initiator, at least one comonomer with reactive
linker groups and, where appropriate, at least one plasticizer.
[0044] After preparing the layer and subsequent thermal or
photocrosslinking, a water-swellable hydrogel is obtained which
contains reactive linker groups for the immobilization of probe
molecules. Crosslinkers which are employed are
methylenebisacrylamide and/or dimethylacrylic esters, for example
tetraethylene glycol dimethacrylate.
[0045] The mesh size of the hydrogel can be adjusted by varying the
concentrations of the crosslinker. The comonomer used contains
maleic anhydride and/or glycidyl (meth)acrylate. Suitable
plasticizers are mono-, di- and/or triethylene glycol. The
reactants mentioned are mixed with a polar, water-miscible solvent,
preferably with dimethylformamide. The processing viscosity can be
adjusted by varying the proportion of the solvent. Adhesion to the
surface of the flat carrier and to the electrode arrangement 5 can
be enhanced by adding customary adhesion promoters, for example
based on silane.
[0046] FIGS. 4 and 5 depict a four-pole electrode arrangement 20.
The electrode arrangement 20 is composed of two current electrodes
22, 23 and two voltage or probe electrodes 24, 25. The current
electrodes 22, 23 are arranged and designed according to the
electrode arrangement 5 of the exemplary embodiment of FIG. 2. The
probe electrodes 24, 25 are likewise strip-like and extend in the
form of a meandering double strand through the spaces between the
partial electrodes 22a and 23a.
[0047] A high-frequency alternating current is applied to the
current electrodes 22, 23. A voltage meter 26 which enables a
change in the electric alternating field as a result of
hybridization events to be detected is connected to the probe
electrodes 24, 25. The measurement can thus be carried out
independently of the current electrodes so that, for example, the
polarization of the latter, which increases the capacitance of the
electrodes, cannot affect the measurement.
[0048] In contrast, in the case of a two-pole electrode
arrangement, electrode capacitance has to be kept low by use of a
correspondingly high measuring frequency unsuitable for the
measurement. This is done in order to be able to determine the
resistance of the analyte solution or of the reaction layer, which
is decisive for the measurement.
[0049] In a variant of the method, depicted diagrammatically in
FIG. 6 (for this and for FIGS. 8 to 10, see the figure legend
indicated further below), an analysis solution 18 which contains a
DNA fragment F.sub.A with a target sequence Z.sub.A, an external
primer pair and the reagents required for a PCR, such as a Taq
(DNA) polymerase, dNTPs (deoxynucleoside triphosphate) etc., is
applied to a microchip 1. The target sequence Z.sub.A is one which
can occur in a plurality of different variants, for example typing
of viruses, e.g. HIV or HPV.
[0050] Each possible variant (Z.sub.A1, Z.sub.A2, etc.) has at
least one separate spot 4A1, 4A2 etc. assigned to it, with a single
oligonucleotide type which can hybridize with a specific target
sequence being immobilized as probe molecule within the reaction
layer 14 of the particular spot. Amplification (PCR) of the target
sequence Z.sub.A, which is carried out in the usual way using a
thermocycling process, takes place only in the mobile phase 18.
[0051] Preference is given to using a primer pair which couples
(hybridizes) outside the target sequence Z.sub.A, as indicated in
FIG. 7. The copied double strand, i.e. strand S.sup.+ and
counterstrand S.sup.-, detach from one another during denaturation
(melting). Normally, the strand S.sup.+ (sense strand) is used for
identifying a target sequence. Accordingly, oligonucleotides which
hybridize exactly with this strand are immobilized in the spots
4A1, 4A2. In the simplest case of the presence of only one DNA
fragment Z.sub.A1, the amplified target sequence Z.sub.A1
accumulates due to hybridization in that spot in which the
correspondingly complementary capture oligonucleotide Z.sub.A1 is
immobilized.
[0052] In the method variant indicated in FIG. 8, the analyte
solution 18 contains various types of DNA fragments. Two of those
DNA fragments, F.sub.A and F.sub.B, are shown by way of example.
One or all of the DNA fragment/s present in the analyte solution 18
may be those according to the method variant of FIG. 6.
[0053] In this case, different groups of spots are to be provided,
with typing of the variants of one DNA fragment being assigned to
each group. However, the analytical investigation may also aim at
"completely" different DNA fragments. In this case, it is
sufficient in principle to assign in each case a single analysis
spot 4A, 4B to a DNA fragment F.sub.A, F.sub.B. As in the method
variant of FIG. 6 too, the analyte solution 18 here contains an
external primer pair. The latter is selected so as to be suitable
for the amplification of all DNA fragments F.sub.A, F.sub.B to be
analyzed (multiplex PCR).
[0054] The capture oligonucleotides indicated in FIG. 6 and FIG. 8
may act as primers in further method variants, if they can be
extended by DNA polymerases. If the reaction layer 18 is permeable
for DNA polymerase and the template/s and the further components of
the PCR reaction, elongation of the immobilized oligonucleotides
takes place according to the sequence of the hybridized matrix.
[0055] In the method variant indicated in FIG. 9, a plurality of
different DNA fragments according to the method variants of FIG. 6
or of FIG. 8 are present. Two of such DNA fragments, F.sub.A and
F.sub.B, are shown by way of example.
[0056] While in the method variants described above a primer pair
was added to the analyte solution, now the solution contains only
one primer of said primer pair in a mobile and dissolved form. This
primer is unspecific, i.e. it is an external primer which couples
to all DNA fragments F.sub.A and F.sub.B present in the analyte
solution outside the target sequence Z.sub.A and Z.sub.B to be
detected (preferably to the sense strand).
[0057] After denaturing of the analyte solution, the DNA single
strands diffuse arbitrarily into the spots. Specific
oligonucleotide capture molecules which bind directly upstream of
the target sequence of the analyte DNA are immobilized in these
spots. A hybridization takes place only where the analyte DNA hits
complementary immobilized oligonucleotides (capture molecules).
[0058] In the subsequent elongation step of the PCR, the 5' end of
the capture oligonucleotides which have previously captured (bound)
selectively the DNA fragments to be detected is extended according
to the information of the hybridized template. The capture molecule
thus becomes the primer for the DNA polymerase reaction. The latter
indicates that elongation can occur only in a microspot which also
contains the capture molecule complementary to the target
sequence.
[0059] In the case of the diagram in FIG. 9, strand S.sup.+ of the
DNA fragment F.sub.A in spot 4A and strand S+of the DNA fragment
F.sub.B in spot 4B are copied by way of elongation of the
particular immobilized primer/capture molecule. In the reannealing
reaction following the particular amplification step and the
melting, the target sequences which were originally present and
those which have been produced in the solution anew accumulate on
complementary capture molecules or sequences.
[0060] Owing to the increase in the concentration of the target
sequence due to the preceding PCR cycle, these sequences will also
bind to capture oligonucleotides which have not yet been extended
and are present in their original form. Thus, the next PCR
elongation step will start from these primers which have been
produced de novo by the hybridization. This results in an increase
in the concentration of extended capture molecules with each PCR
cycle. This increase in the concentration of capture
molecules/primer extended by about 100-300 bases (compared to 20-30
bases originally) causes a change in the electrical field or
resistance, which may be measured with the aid of the electrode
arrangement 5 or 20 and utilized for PCR monitoring (on-line
PCR).
[0061] In the method variant of FIG. 10, the analyte solution 18
contains one or more DNA fragment species according to the method
variants of FIG. 6 or FIG. 8. While in the variants of FIG. 6 and
FIG. 8 primer pairs and in the variant of FIG. 9 only one primer of
the primer pair/primer pairs were added to the analyte solution,
the solution here does not contain any dissolved free primers. The
elongation reactions here take place in the individual microspots
4A, 4B, etc.
[0062] In contrast to all other method variants, here (FIG. 10)
both capture molecules of the capture molecule pair which are
needed for the specific detection of the two DNA strands of the
target sequence are immobilized in each case in the same gel spot,
i.e. an immobilized internal capture molecule/primer pair which
hybridizes to a target sequence is present. After melting
(denaturing) of the sample, the DNA single strands diffuse randomly
into the spots. The strand S.sub.A.sup.+ or S.sub.A.sup.-
hybridizes with its 5' or 3' end to the complementary primer of the
primer pair in spot 4A.
[0063] According to FIG. 10, the S.sup.+ strand of the DNA fragment
F.sub.A binds to a primer indicated by A.sup.+ and the S.sup.-
strand of the DNA fragment F.sub.A binds to a primer indicated by
A.sup.-, in each case at spot 4A. Subsequently, an antiparallel
strand S.sup.- or S.sup.+ is formed which is then likewise
immobilized, since it has been synthesized by way of elongation of
the immobilized primer A.sup.+ or A.sup.-.
[0064] In contrast to this, the 3' end of the elongated strand
S.sup.- or S.sup.+ moves freely and can hybridize with a
counterprimer A.sup.- or A.sup.+ immobilized in its proximity in
the reaction layer. From this, the following amplification step
results in an S.sup.+ or S.sup.- strand which is likewise
immobilized since it was formed by way of elongation of the
immobilized primer A.sup.+ or A.sup.-.
[0065] The increase in the concentration of appropriately extended
capture molecules, which occurs in each PCR cycle, causes, as has
been described already further above, a change in the electric
field or resistance. This may be utilized with the aid of the
electrode arrangement 5 or 20 for PCR monitoring, thus making
online PCR possible.
[0066] The novel method for amplification and detection of
nucleotide sequences, which has been described in detail above,
uses a biochip as described in the alternatives as a two-pole or
four-pole arrangement in FIGS. 2 and 4.
[0067] It is apparent, especially from FIGS. 2 and 4, that the
biochip suitable for the method described, including the reaction
layer arranged thereupon and the analyte solution in contact
therewith, is arranged in a housing depicted as a frame in said
figures. According to FIGS. 6 and also 8 to 10, the housing is open
at the side so that the analyte solution can flow through past the
reaction layer.
[0068] Legend of the symbols in the above description and the
figures:
.uparw.=primer, in solution
x=unspecific
A,B=relating to a particular DNA fragment
.perp.=immobilized oligonucleotide, without primer function
.quadrature.=immobilized primer
F=DNA fragment
S=single strand of a DNA fragment
.sup.+/.sup.-=relating to a coding/noncoding strand
[0069] Example embodiments of the present invention being thus
described, it will be obvious that the same may be varied in many
ways. Such variations are not to be regarded as a departure from
the spirit and scope of the present invention, and all such
modifications are intended to be included within the scope of the
present invention.
* * * * *